Project Summary The proteasome plays a significant role in the proper functioning of eukaryotic cells. It is responsible for up to 90% of the cell's protein degradation needs, controls cell pathways with the proper balance of critical protein levels, and produces antigenic peptides to allow the immune system to recognize diseased cells. To meet these diverse needs of the organism, different forms of the proteasome exist with corresponding specialties. There are limited tools available to analyze the different types of proteasome isoform activities in the cells. Proteasome mediated protein hydrolysis can occur through at least three different paths. The tools currently available cannot effectively differentiate between the activity of these proteasome isoforms and are easily hydrolyzed by other proteases in the cell. The goal of this project is to design proteasome isoform-selective activity probes. We will extensively utilize the data accumulated to produce selective inhibitors to design our activity probes. Upon completion of this project, we will have new proteasome activity probes that can differentiate the activity of ubiquitin-dependent and -independent degradation. We will also have a probe that can detect protein hydrolysis mediated by the immunoproteasome only. The design of our probes will also include significant secondary structure and peptidomimetic subunits to prevent non-specific hydrolysis by proteases in cells. Distinguishing the activity of the types of proteasomes within a cell would clarify which protein degradation pathway could be targeted as a new therapy. We anticipate they can be used to monitor the amount of ubiquitin- dependent and -independent degradation in protein accumulation diseases and in hematological cancers. The immunoproteasome has recently been implicated in a variety of autoimmune diseases and type I diabetes. Our probes will provide a way to determine how much immunoproteasome activity affects these diseases. We believe these new probes will fill an empty niche in the proteasome field for when one wants to study the activity of an individual proteasome isoform.